Process for the manufacture of the corticotropic hormone



Patented Deo. 28, 1943 PROCESS FOR THE MANUFACTURE CORTICOTROPIC HORMONEor run Karl Junkmann, Berlin, Germany, asslgnor to sobering Corporation,Bloomfield, N. 1., a corporatlon of New Jersey No Drawing. ApplicationJune as, 1939, Serial No. 281,632. In Germany July 29, 1938 1 Claim.(Cl. 167-74) This invention relates to a Pr ss 1' e manufacture or thecorticotropio hormone.

The anterior pituitary lobe contains a hormone which is capable ofrectifying changes caused in the suprarenal cortex on removal of thepituitary. as for example the occurrence of a zone containing less fat(sudanophobe) (Collip and others,

Lancet, 1933 page 347; Nirehours Archiv vol. 290,

page 23, 1933). For production of this hormone there has been employedultra-filtration (Hofimann and Anselmino Archiv f. Gynakologie, vol.157, page 86, 1934) or separation by alcohol (Dutch Patent 77,332).These prior methods, however, as we have ascertained by testing them,only yield the hormone in a very impure state.

In accordance with the present invention the hormone is obtained in apure state and in good yield by the application of a method involving acombination of precipitation steps. In the method of the invention theaqueous extract of the anterior pituitary lobe produced by extractionwith weakly acid or alkaline media at elevated temperature, suitably atboiling temperature in known manner, is freed from albuminous substancesby means of a precipitating agent, such as sulpho-salicylic acid, or byany other a1- bumen-precipitating agent whose capacity for precipitatingalbumens while leaving the corticotropic hormone in solution has beenpreviously established by test experiments, preferably at strong acidreaction for example pH=1 to 2. From the albumen-free extract thehormone is then precipitated by means of a precipitating agent of thetype disclosed hereinbelow, or by other agents capable of precipitatingthe hormone without destroying its therapeutic activity, as can bedetermined by simple experiment. The precipitate obtained is freed inknown manner from precipitatin agent, suitably by dissolving in dilutealkali and precipitating by means of alcohol or acetone or any suitablewater-soluble solvent.

As albumen precipitating agent sulpho-salicylic acid has proved to beparticularly suitable, while for precipitation of the hormone,precipitating agents such as picric acid, picrolonic acid, flavianicacid, phosphotungstic acid and others can be employed. Sulphosalicylicacid and trichloraoetic acid are unsuitable for the step ofprecipitating the hormone.

It is obvious that other precipitating agents than those specificallydisclosed in the foregoing may be used to obtain the corticotropichormone.

The suitability of such precipitating agents must,

of course, be determined beforehand by tests as to their precipitatingaction and by biological tests of the products obtained. The biologicaltests consist in determining the histological changes produced in thesuprarenal cortex of hypophysectomized rats upon administration oi theproducts. These biological tests are described in detail in BomskovMethoden der Hormonforsohung, vol. 2 (1939), pages 965-971, and inAbderhaldens "Handbuch der biologischen Arbeitsmethoden, sec. V, part33, 2nd half, pages 1107-1108, 1938.

The pure hormone is a white, water-soluble powder of good activity whichis free from glandular tissue, from the naturally accompanyingalbuminous substances, and from other hormones of the anterior pituitarylobe.

The following example illustrates the invention:

Examples 1 kg. of the de-fatted dry powder from acetone treated anteriorpituitary lobes of cattle is extracted at boiling temperature withstirring for 30 minutes with 20 litres of /100 N HCl. The centrifuged orpressed oif extract is acidified with HCl to pH 2 and treated with thenecessary quantity, determined in a preliminary experiment, of a 20%aqueous solution of sulpho-salicylic acid. The quantity ofsulpho-salicylic acid is calculated on the basis that in the testfiltrate on further addition no more turbidity is produced. Theseparated precipitate is brought into solution in 5 litres of water withthe necessary quantity of concentrated ammonia and again precipitated byfurther addition of sulpho-salicylic acid. The filtrate therefrom iscombined with that from the first sulphosalicylic acid precipitate andafter addition of sufiicient caustic soda solution to reduce the mixtureto a weakly acid reaction to Congo red, the hormone is precipitated bysaturation with picric acid. The picric acid precipitate; centrifuged orfiltered oil! is treated in 500 cc. of water with the necessary quantityof ammonia to a condition neutral to litmus, whereby the precipitate forthe most part passes into solution. From this solution the hormone isprecipitated by addition of 5-10 times the volume of alcohol or acetoneand dried with ether. Yield 8.0-10.0 grams which contain -80% of thetotal activity of the starting material. If a unit is taken as thatquantity to be injected daily, which on 7 days continuous dailyinjection into hypophysectomised rats, is Just capable of causing thesudanophobe zone in the suprarenal cortex to disappear, then thepreparation obtained acoordins to the invention contains pergram ofstartina' material 1 million to 1.2 millions units.

The therapeutic usefulness of corticotropic hormone is already known.Thus Griineberg,

Archiv tiir Dermakologie und Syphilis (1937); vol. 174, page-638,reports good results in the treatment of psoriasis with corticotropichormone. The treatment is given intramuscularly or subcutaneously over aperiod of two to three A process for the production of the corticotropichormone, which comprises extracting defatted anterior pituitary lobeswith dilute hydrochloric acid, clarifying the extract, addinghydrochloric acid to a pH of about 2, adding an aqueous solution ofsulphosalicylic acid to precipitate albumens, removing the precipitate,treating the solution with alkali to render the same-weakly I acid toCongo red, then treating the resulting solution with a member of thegroup consisting of picric, picrolonic, flavianic, and phosphotungsticacids, dissolving the hormone precipitate with such an amount of alkalias to yield a solution which is neutral to litmus, and precipitatin thehormone by the addition of a water-miscible organic solvent.

KARL JUNKMANN.

